The human immunodeficiency virus type I (HIV- 1) genome encodes the regulatory protein Tat (transacting transcription factor) which is a powerful trans-activator of gene expression at one or more levels and is required for efficient virus growth. Tat is 86 amino acid long, contains a cysteine rich region (from residue 22 to 37) which enables Tat to form a metal linked dimer, and it also includes a highly basic region (from residue 49-60) which is characteristic of some nuclear I DNA binding proteins. During the past year, we have been able to express Tat protein in E. Coli cells by using a unique E.Coli expression system in which the Tat protein coding region was fused to the coding region for the IgG binding domain of protein A (designated Z). The coding region was altered so that chemical cleavage could be achieved between the Tat protein and Z-Z- polypeptide. By using IgG-Sepharose, gel-filtration (HPLC) chromatography, we have been successful in obtaining 15 mg of purified Tat protein. Sodium dodecyl sulfate gel electrophoresis reverse phase HPLC, amino acid composion analysis, amino-terminal polypeptide sequencing was used for biochemical characterization. Biological or functional activity was assayed using an in vitro transcription system. Polyclonal antibodies were generated against three synthetic peptides (including regions 1-22, 53-72, 73-86 of the HIV-Tat protein sequence) and full-length recombinant Tat protein. AH antibodies were successful in blocking Tat trans-activation of the HIV-LTR promoter in the in vitro transcription assay. Furthermore anti-peptide(53-72) cross reacted more strongly with the Tat protein than the other two peptides suggesting that the 53-72 region may be on the surface of the Tat molecule. We are attempting to refold the Tat protein with thiol reagents and varying renaturing conditions to facilitate the study of the biologically relevant three-dimensional conformation.